S-100B is used in melanoma follow-up. This serum biomarker is also present in adipocytes; therefore, subcutaneous adipocytes trapped in the needle before performing a venipuncture could contaminate the serum. The aim was to study the influence of adipocyte contamination on blood samples used for S-100B analysis, possibly resulting in falsely elevated S-100B values. A total of 294 serum samples were collected from 147 American Joint Committee on Cancer staging stage III melanoma patients. The mean difference between the first (dummy) and second tubes was 0.003 μg/l (p = 0.077), with a decrease in the second tube. Compared with the second tube, the S-100B level was higher in the first tube in 33.3% of the samples, equal in 36.8% of the samples and lower in 29.9% of the samples. No significant difference between the two consecutively drawn tubes was found. There seems to be no necessity of implementing a dummy tube system for accurate S-100B determination in melanoma patients.